The Regulatory Effect Of TFEB On The Autophagy Induced By Methionine Restriction In SH-SY5Y Cells

Background: Studies in many organisms have shown that methionine restriction is a potential anti-aging intervention to prolong the lifespan.However,its mechanisms have not been well explored.Autophagy,a clearing mechanism for intracellular damaged substances,plays an important role in regulating aging process.Transcription factor EB(TFEB)can regulates autophagy at the transcriptional level.Evidence suggested that methionine restriction can exert its anti-aging effect by affecting autophagic function.Further exploring the effect of methionine restriction on autophagic pathway and underlying TFEB-mediated will help to broaden our understanding of anti-aging mechanisms of methionine restriction and provide scientific basis for its application in eldly population.Objective: 1)To determine whether methionine restriction can induce autophagy.2)To explore the effect of methionine restriction on lysosomal activity.3)to explore the effects and mechanisms of TFEB in autophagy induced by methionine restriction.Methods: 1)Methionine restriction: SH-SY5 Y cells were incubated in control,50%,75%,87.5% and 93.75% methionine restriction medium for 24 hours.2)A CCK-8 cell viability assay kit was used to detect the survival rate of SH-SY5 Y cells after methionine restriction treatment.3)After methionine restriction treatment for 24 hours,SH-SY5 Y cells were incubated with 40 m M chloroquine(CQ)for 2 additional hours,and antophagy-related proteins levels in cells treated with/without CQ and the protien levels of TFEB in nuclear extract were detected by western blot.4)Quantitative PCR was used to measure the m RNA levels of TFEB-target genes in SH-SY5 Y cells treated with methionine restriction for 24 hours.5)Lysosomes in SH-SY5 Y cells treated with methionine restriction were stained with Lyso-Tracker Red and observed using a fluorescence microscopy.6)TFEB expression in SH-SY5 Y cells were knocked down using TFEB si RNA interference,and then cells were treated with 87.5% methionine restriction for 24 h,and antophagy-related proteins levels in cells treated with/without CQ autophagy-related proteins were detected by western blot.7)and the lysosomes were stianed with Lyso-Tracker Red and observed using a fluorescence microscopy.Results: 1)As compared with control,50%,75%,and 87.5% methionine restriction did not affect SH-SY5 Y cell viability;while the cell viability was significantly decreased by 93.75% methionine restriction(p<0.05).2)methionine restriction at 50%,75%,87.5% and 93.75% levels decreased protien levels of autophagy-related proteins LC3Ⅱ,Beclin-1and SQSTM1/p62 after 24 hours treatment(p<0.05);Additional incubation with 40 m M CQ for 2 hours resulted in increased levels of these proteins(p<0.05).3)LAMP1 protien levels were increased by 50%,75%,87.5%and 93.75% methionine restriction treatment,which was not modified by additional CQ incubation.4)The amount of lysosones stained by Lyso-Tracker Red in 50%,75%,87.5% and 93.75% methionine restriction treated cells were significantly increased.5)TFEB protein levels in nuclear extract of cells treated with 50%,75%,87.5% and 93.75%methionine restriction were significantly increased,as comapared with control cells;Meanwhile,m RNA levels of autophagy-lysosome related genes including TFEB,LC3,SQSTM1/p62,LAMP1 and CTSA,which were also TFEB-regulated genes,were significantly induced by methionine restriction treatment.6)Knockdown of TFEB expression using TFEB si RNA interference abrogated autophgy induced by methionine restriction and lysosome generation expression..Conclusion: Methionine restriction induces autophagy in SH-SY5 Y cell,which was mediated by TFEB.TFEB plays a critical role in mediating autophagy induced by methionine restriction and substrate degradation in lysosome.